Work in this proposal addresses the Program Announcement (PA-06-525) entitled "MDMA: Research Areas Needing More Emphasis (R21)". The most dangerous side effect of acute MDMA intoxication is hyperthermia, a hyper-metabolic condition that stems from a dramatic increase in "non-shivering" metabolic thermogenesis in the setting of heat conservation (peripheral vasoconstriction). MDMA-induced hyperthermia leads to a cascade of life threatening medical complications including skeletal muscle breakdown, cardiovascular collapse, multi-organ failure, and disseminated intravascular coagulation. In animals, hyperthermia also strongly potentiates long term neurotoxicity. No treatment exists for the reversal of hyperthermia induced by MDMA and related compounds, most likely because the direct mediators of human thermogenesis in response to normal (cold, feeding, infection) and pathologic (MDMA, endocrinopathies) thermogenic stimuli have not been identified. Uncoupling proteins (UCPs) are highly conserved thermogenic molecules that regulate mitochondrial heat production in plants, nematodes, flies, and vertebrates. In mammals, brown fat UCP1 is thought to be the dominant, if not the only significant mediator of thermogenic responses. Arguing strongly against this is the fact that adult humans express negligible amounts of brown fat and UCP1, yet do show significant non-shivering thermogenic responses (i.e. MDMA hyperthermia, diet induced thermogenesis, fever). Skeletal muscle is the most important thermogenic organ in humans, and a major target of MDMA-induced pathology;thus, we reasoned that the recently identified uncoupling protein 3, expressed primarily in skeletal muscle (UCP3) may mediate MDMA hyperthermia. We tested this in mice lacking UCP3. UCP3 knockout mice have a near complete loss (~80%) of MDMA-induced thermogenesis and are completely protected from MDMA-induced death over a range of lethal doses. Consistent with the general thermoregulatory relevance of UCP3 in inducible thermogenesis, we also observed that UCP3 knockout mice lose the capacity to generate a fever in response to the bacterial pyrogen lipopolysaccharide. With regard to mechanisms of UCP3 activation, we observed that free fatty acids - required ligands for UCP3 function in vitro - are elevated in the bloodstream prior to peak MDMA-induced hyperthermia. Additionally, we found that high fat feeding increases levels of plasma free fatty acids and correspondingly potentiates MDMA hyperthermia. The overall goal of this proposal is to identify the tissue target(s) and mechanisms involved in MDMA-induced UCP3-dependent thermogenesis. Specific Aim #1: To identify the target tissue of UCP3-dependent thermogenesis. Specific Aim #2: To characterize the role of free fatty acids in UCP3 activation by MDMA in vivo. Together, these studies will significantly advance our mechanistic understanding of this novel, UCP3-dependent thermogenic pathway in humans and may reveal novel drug targets for the treatment of a range of thermoregulatory illnesses ranging from hyperthermia to obesity and diabetes. Work in this proposal addresses the program announcement PA-06-525, entitled "MDMA: Research Areas Needing More Emphasis". One of the most widely abused drugs in the world, MDMA overdose can give rise to fatal hyperthermia. No treatment exists for MDMA hyperthermia. Work in this proposal aims to determine where in the body heat generation occurs after MDMA treatment in animals, and the molecules that are involved in generating the heat response. It is expected that this work will provide a rational basis for the development of the first effective hyperthermia medicine able to directly turn off heat production.